Microorganism producing 5&#39;-inosinic acid and process for producing 5&#39;-inosinic acid using the same

ABSTRACT

The present invention relates to a novel microorganism,  Corynebacterium ammoniagenes  strain CJIP009 having Accession No. KCCM-10226, which is capable of producing 5′-inosinic acid and a process for producing 5′-inosinic acid using the same.

[0001] This application is a continuation application of InternationalApplication Serial No. PCT/KRO1/02264, filed Dec. 26, 2001 and publishedin English as WO 02/051984, which claims priority to Korean ApplicationSerial No. 2000/81471, filed Dec. 26, 2000. Said applications areincorporated herein in their entirety by reference.

TECHNICAL FIELD

[0002] The present invention relates to a novel microorganism producing5′-inosinic acid and to a process for producing 5′-inosinic acid usingthe same.

BACKGROUND

[0003]5′-inosinic acid is an intermediate material of the metabolicsystem of nucleic acid biosynthesis, which is used in a variety offields, like foods and medicines, and in various kinds of medical areasand is important in animal and plant physiology. In particular,5′-inosinic acid is a nucleic acid-type seasoning that has synergiceffect when used with sodium glutamate.

[0004] Processes for producing 5′-inosinic acid by direct fermentationhave been known in this field, and the important key in economicalaspects was to produce 5′-inosinic acid in a high concentration andyield.

DISCLOSURE OF THE INVENTION

[0005] The present inventors conducted extensive studies to develop anew strain capable of achieving the above mentioned purposes, and as aresult, discovered a novel microorganism producing 5′-inosinic acid bydirect fermentation in a high concentration and yield.

[0006] The present invention provides a mutant of Corynebacteriumammoniagenes CJIP009 (Accession Number KCCM-10226) that is characterizedby accumulating 5′-inosinic acid in a high concentration and yield bydirect fermentation and having resistance to L-glutamine analoguesselected from Azaserine or 6-diazo-5-oxo-L-norleucine (DON), andresistance to L-proline analogues selected from 3,4-dehydroproline,L-azetidine-2-carboxylic acid, L-thiazolidine-4-carboxylic acid,(S)-2,2-dimethyl-4-oxazolidecarboxylic acid,(S)-5,5-dimethyl-4-thiazolide carboxylic acid,(4S,2RS)-2-ethyl-4-thiazoline-carboxylic acid,(2S,4S)-4-hydroxy-2-pyrroline-carboxylic acid, 2-piperidinecarboxylicacid or 2,5-pyrrolidinedione.

[0007] The present invention also provides a process for producing5′-inosinic acid characterized by cultivating the mutant ofCorynebacterium ammoniagenes CJIP009 (KCCM-10226) followed by collectionof the cultivated substances.

[0008] The artisan of ordinary skill will recognize that Corynebacteriumammoniagenes was formerly called Brevibacterium ammoniagenes. See e.g.Collins, 1987, “Transfer of Brevibacterium ammoniagenes (Cooke andKeith) to the genus Corynebacterium as Corynebacterium ammoniagenescomb. nov.”, Int. J. Syst. Bacteriol. 37:442-443.

[0009] The microorganism of the present invention, a mutant ofCorynebacterium ammoniagenes (ATCC-6872), requires Adenine, but does notrequire Xanthine or Guanine. However, growth of the mutant microorganismof the invention is facilitated by adding Xanthine or Guanine, whencompared with a conventional Adenine Leaky Mutant producing 5′-inosinicacid [Agr. Bio. Chem., Vol. 47(5), pp. 1035-1041, 1983, (KY13102,KY13171, KY13184, etc.)] The mutant microorganism of the invention maysimultaneously require Adenine and Xanthine or Guanine.

[0010] In addition, the microorganism of the present invention lacksUrease to assimilate Urea, and has a high sensitivity to lysozyme, thecell wall degrading enzyme, which is considered that the capacity ofcell wall synthesis is partially lost, so that lots of intracellularlyproduced 5′-inosinic acid is easily secreted out of the cell. Accordingto the invention, a microorganism is sensitive to lysozyme if theminimum inhibitory concentration is equal to or less than 8.0 μg/mL.

[0011] Balabushevich, M. I. and Kazarinova, L. A., et al (Prikl.Biokhim. Mikrobiol., 19(5), 590-598, 1983), Russia, discovered thatadding streptomycin and kanamycin to the medium enhanced cell wallpermeability and helps accumulation of 5′-inosinic acid in the medium.Considering this, it was hypothesized that a mutant, capable ofproducing 5′-inosinic acid in a high concentration and yield, would havebeen discovered by introducing resistance of streptomycin to a knownstrain to enhance the membrane permeability of the microorganism. Then,it was further hypothesized that the contamination frequently occurringin fermentation would be prevented by using the above mutant and addingthe streptomycin to the medium. Therefore, the present inventorsobtained a strain having resistance to a high concentration ofstreptomycin and studied properties of the strain. In practice, it wasdetermined that strains having resistance to high concentrations ofstreptomycin may be grown without contamination occurring duringfermentation.

[0012] Most bacteria accumulate calcium ions and organic solutes, i.e.,osmolytes, by improving intracellular osmotic pressure of bacteria toprevent osmotic dehydration under extracellular osmotic pressure ofbacteria. Such osmolytes include L-proline, L-glutamate, sugar,N-methylated amino acid derivatives, etc. Among these, L-proline hasbeen known as an important factor of osmoregulation. It has beenreported that intracellular L-proline accumulated in Brevibacteriumtyphimurium by increasing the activity of pyrroline-5-carboxylatereductase, which is the important enzyme of biosynthesis pathway ofproline when the extracellular concentration of 5′-inosinic acidincreased [Agr, Bio, Chem., Vol. 53(9), pp. 2475-2479, 1989]. Inaddition, it was reported that extracellular osmotic pressure inducedintracellular accumulation of L-proline in Escherichia coli, Salmonellatyphimurium, Serratia marcescens, and others [J, Bacteriol., Vole 163,p296, 1985].

[0013] Accordingly, it is contemplated that in order to have amicroorganism capable of producing 5′-inosinic acid in a highconcentration and yield, it is important to prevent the inhibition ofgrowth and biochemical metabolic process by increasing intracellularL-proline synthesis and that it is important to reinforce the osmoticpressure-resistant characteristics by enhancing the synthetic capacityof L-proline.

[0014] Further, in order to produce 5′-inosinic acid from5-phosphoribosyl-α-1-pyrophosphate (PRPP), i.e., a purine-type nucleicacid precursor, 2 molecules of glutamine are needed. Glutaminesynthetase, which produces glutamine from sodium glutamate, is veryelaborately regulated by amino acids, such as glycine, alanine,histidine, etc., and CTP, AMP, etc. [Escherichia coli and Salmonellatyphimurium, 1987, p302˜320]. Therefore, a harmonious supply ofglutamine is necessary for the synthesis of 5′-inosinic acid. Further,produced glutamine is widely used as a precursor of various reactions.In the synthesis of 5′-inosinic acid, two enzymes, which are PRPPamidotransferase and 5-phosphoribosyl-N-formylglycinamide (FGAR)amidotransferase, employ glutamine as a substrate [Escherichia coli andSalmonella typhimurium, 1987, pp. 445-473]. Therefore, in order to moreeffectively achieve 5′-inosinic acid synthesis, it is considered thatthe harmonious synthesis of glutamine is important to increase theaffinity of PRPP amidotransferase and FGAR amidotransferase related tosynthesis of 5′-inosinic acid to the glutamine rather than other enzymesamong various reaction requiring glutamine.

[0015] Accordingly, the present inventors tested the effect of variousamino acids on the synthesis of 5′-inosinic acid by direct fermentation.As the result, the inventors determined that the fermentationconcentration of 5′-inosinic acid increased upon addition of L-glutamineto the medium. The inventors further determined that the appropriatesupply of L-glutamine to the present strain is a rate-limiting step inthe synthesis of 5′-inosinic acid.

[0016] The inventors identified that the microorganisms introducedresistance to L-glutamine analogues, such as Azaserine or6-diazo-5-oxo-L-norleucine (DON), and resistance to various L-prolineanalogues, such as 3,4-dehydroproline, L-azetidine-2-carboxylic acid,L-thiazolidine-4-carboxylic acid, (S)-2,2-dimethyl-4-oxazolidecarboxylicacid, (S)-5,5-dimethyl-4-thiazolide carboxylic acid,(4S,2RS)-2-ethyl-4-thiazoline-carboxylic acid,(2S,4S)-4-hydroxy-2-pyrroline-carboxylic acid, 2-piperidinecarboxylicacid or 2,5-pyrrolidinedione can produce 5′-inosinic acid by directfermentation method in a higher concentration and yield than the knownstrains. The present invention is based on such discovery.

[0017] The microorganism of the present invention, a mutant ofCorynebacterium ammoniagenes (ATCC-6872), requires Adenine, but does notrequire Xanthine or Guanine, though the growth is facilitated by addingthem. In addition, the microorganism lacks Urease to assimilate Urea andis highly sensitive to lysozyme. Without being restricted to aparticular model, it is believed that the mutant microorganism of theinvention is partially deficient in cell wall synthesis, a propertywhich may promote secretion of intracellularly produced 5′-inosinicacid. The microorganism of the invention may be resistant tostreptomycin, particularly high concentrations of streptomycin.

[0018] A high concentration of glucose or other carbon sources addedduring the culture period and 5′-inosinic acid accumulated during thelater culture period lead to an increase in extracellular osmoticpressure of 5′-inosinic acid-producing microorganisms thereby inhibitingtheir normal physiological activities and cell growth. Therefore, it isdesirable to improve the osmotic resistance to prevent the reduction of5′-inosinic acid production.

[0019] To increase the intracellular concentration of proline whichplays an important role in osmoregulation to a high accumulation ofsolutes in the extracellular environment, the microorganism of thepresent invention has resistance to L-proline analogues, such as3,4-dehydroproline, L-azetidine-2-carboxylic acid,L-thiazolidine-4-carboxylic acid, (S)-2,2-dimethyl-4-oxazolidecarboxylicacid, (S)-5,5-dimethyl-4-thiazolide carboxylic acid,(4S,2RS)-2-ethyl-4-thiazoline-carboxylic acid,(2S,4S)-4-hydroxy-2-pyrroline-carboxylic acid, 2-piperidinecarboxylicacid or 2,5-pyrrolinedione, thereby excluding the effect of osmoticpressure more efficiently.

[0020] Further, the microorganism of the present invention hasresistance to L-glutamine analogues, such as Azaserine or6-diazo-5-oxo-L-norleucine (DON), essentially required in thepurine-type synthesis system by achieving the harmonious supply ofglutamine for the synthesis of 5′-inosinic acid, resulting in directlyaccumulating 5′-inosinic acid in a high yield and concentration.

[0021] The protocol by which the mutant microorganims of the presentinvention was isolated began with strain CJ 12 as the parental strainand consisted of several iterations of mutagenesis and screening. Ineach iteration, microorganisms were treated with X-ray irradiation,ultraviolet ray irradiation, and/or a chemical mutagen, such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG), diethyl sulfate, ethylamine, etc., and then screened for 5′-inosinic acid production. Treatedbacteria were suitably suspended and spread on a minimal medium (Medium2) containing 1.7% agar and each concentration of variants. Then, eachcolony was cultivated on a nutrition medium (Medium 1) followed by aseed medium (Medium 3) for 24 hours and a fermentation medium (Medium 4)for 5-6 days. Colonies that produced the highest levels of 5′-inosinicacid were selected for subsequent rounds of mutagenesis.

[0022] After several iterations this mutagenesis protocol, the inventorsisolated the microorganism of the present invention, which produces highlevels of 5′-inosinic acid, lacks Urease, is sensitive to lysozyme, isresistant to L-glutamine, L-proline, and high concentrations ofstreptomycin, and requires Adenine, but does not require Xanthine orGuanine, though growth is facilitated by adding them, and lacks Ureaseto assimilate Urea. This strain was designated CJIP009. Corynebacteriumammoniagenes CJIP009 was deposited under the Budapest Treaty to theKorean Culture Center of Microorganisms whose address is Hongje-dong,Seodaemun-gu, Seoul 120-749, on Nov. 15, 2000 and assigned Accession No.KCCM-10226.

[0023] Specifically, the present invention provides a process forproducing 5′-inosinic acid by cultivating Corynebacterium ammoniagenesstrain CJIP009 (KCCM-10226) on a seed medium at 30° C. for 24 hours,cultivated and activated on a fermentor seed medium at 28-34° C., 900rpm and pH 7.2 for 1-2 days, cultivated on a fermentor main medium at30° C., 900 rpm and pH 7.2 for 5-6 days. When reducing sugars arepresent in the culture solution at a concentration of 2% (w/v), amixture of fructose, glucose, and molasses was added four times untilthe final concentration of reducing sugars in the culture solution wasincreased to 32% (w/v).

[0024] Culture media employed in the present invention have thefollowing compositions:

[0025] Medium 1: Nutrition Medium

[0026] peptone 1%, Beef extract 1%, Sodium Chloride (NaCl) 0.25°/a,Yeast Extract 1%, Agar 2%, pH 7.2

[0027] Medium 2: Minimal Medium

[0028] Glucose 2.0%, Ammonium Sulfate ((NH4)₂SO₄) 0.3%, PotassiumDihydrogen Phosphate (KH₂PO₄) 0.1%, Potassium Monohydrogen Phosphate(K₂HPO₄) 0.3%, Magnesium Sulfate (MgSO₄-7H₂O) 0.3%, Calcium chloride(CaCl₂) 10 mg/L, Ferric Sulfate (FeSO₄.7H₂O) 10 mg/L, Zinc Sulfate(ZnSO₄.7H₂O) 1.0 mg/L, Manganese Chloride (MnCl₂.H₂O) 3.6 mg/L,L-Cystein 20 mg/L Calcium Pantothenate 10 mg/L, Thiamine.HCl 5.0 mg/L,Biotin 30 μg/L, Adenine 20 mg/L, Guanine 20 mg/L, pH 7.3

[0029] Medium 3: Seed Medium

[0030] Glucose 5%, peptone 0.5%, Beef extract 0.5%, Yeast Extract 1%,Sodium Chloride (NaCl) 0.25%, Adenine 100 mg/L, Guanine 100 mg/L, pH 7.2

[0031] Medium 4: Flask Fermentation Medium

[0032] Sodium Glutamate 01%, Ammonium Chloride (NH₄Cl) 1.0%, MagnesiumSulfate (MgSO₄.7H₂O) 1.2%, Calcium Chloride (CaCl₂) 0.01%, FerricSulfate (FeSO₄.7H₂O) 20 mg/L, Manganese Sulfate (MnSO₄.H₂O) 20 mg/L,Zinc Sulfate (ZnSO₄.7H₂O) 20 mg/L, Cupric Sulfate (CuSO₄.7H₂O) 5.0 mg/L,L-Cystein 23 mg/L, Alamne 24 mg/L, Nicotinic acid 8.0 mg/L, Biotin 45μg/L, Thiamine.HCl 5.0 mg/L, Adenine 30 mg/L, phosphoric acid(H₃PO₄)(85%) 1.9%, the mixture of Fructose, Glucose and molasses to 8%(w/v) as reducing sugar (pH 7.2).

[0033] Medium 5: Fermentator Seed Medium

[0034] Glucose 5.4%, peptone 1.0%, Yeast Extract 2.0%, PotassiumDihydrogen Phosphate (KH₂PO₄) 0.1%, Potassium Monohydrogen Phosphate(K₂HPO₄) 0.1%, Magnesium Sulfate (MgSO₄.7H₂O) 0.1%, Ammonium Sulfate((NH4)₂SO₄) 0.5%, Ferric Sulfate (FeSO₄.7H₂O) 80 mg/L, Zinc Sulfate(ZnSO₄ 7H20) 40 mg/L, Manganese Sulfate (MnSO₄.H₂O) 40 mg/L, L-Cystein80 mg/L, Calcium Pantothenate 60 mg/L, Thiamine-HCl 20 mg/L, Biotin 240μg/L, Adenine 1200 mg/L, Guanine 1200 mg/L (pH 7.2).

[0035] Medium 6: Fermentor Main Medium

[0036] Calcium Chloride (CaCl₂) 120 mg/L, Cupric Sulfate (CuSO₄.7H₂O)8.0 mg/L, Magnesium Sulfate (MgSO₄.7H₂O) 1.5%, Ferric Sulfate(FeSO₄.7H₂O) 24 mg/L, Zinc Sulfate (ZnSO₄.7H₂O) 24 mg/L, ManganeseSulfate (MnSO₄.H₂O) 24 mg/L, L-Cystein 26.4 mg/L, Sodium Glutamate0.12%, Thiamine.HCl 6.0 mg/L, Biotin 40 μg/L, Nicotinic acid 50 mg/L,Alanine 145 mg/L, Adenine 200 mg/L, phosphoric acid (H₃PO₄)(85%) 4.3%,the mixture of Fructose, Glucose and molasses to 32% as reducing sugar(pH 7.2)

[0037] The biochemical properties of representative variants of thenovel strain CJIP009 according to the present invention is shown in thefollowing Table 1 (This invention is not limited to the followingproperties). TABLE 1 CJIP009 Property ATCC6872 (KCCM-10226) Adenine Notrequire Require Guanine (Xanthine) Not require Leaky Sensitivity tolysozyme (minimal   80 μg/ml     8 μg/ml  growth inhibitionconcentration) Resistance to 3,4-dehydroproline 1,000 μg/ml  3,500μg/ml  Streptomycin   500 μg/ml  2,000 μg/ml  L-azetidine-2-carboxylicacid    5 mg/ml   30 mg/ml L-thiazolidine-4-carboxylic acid   10 μg/ml   100 μg/ml  Azaserine   25 μg/ml    100 μg/ml 

[0038] The culture process of 5′-inosinic acid used in the invention wasas follows.

[0039] The microorganisms, belonging to Corynebacterium genus andcapable of producing 5′-inosinic acid, were cultured in conventionalmedium containing carbon sources, nitrogen sources, amino acids,vitamins, etc., under aerobic condition with regulated temperature, pH,etc.

[0040] Glucose, fructose, sterilized pre-treated molasses (molassesreverted to reducing sugar) and so on, would be used as a carbon source.Among inorganic nitrogen sources such as, ammonia, ammonium chloride,ammonium sulfate and organic nitrogen sources such as, peptone,NZ-amine, meat extract, yeast extract, corn digestive solution, caseinhydrolysate, fishes or degradation products thereof, de-fatted soybeancake or degradation products thereof and so on, each would be used as aorganic nitrogen source. Potassium Dihydrogen Phosphate (KH₂PO₄),Potassium Monohydrogen Phosphate (K₂HPO₄), Manganese Sulfate(MnSO₄.H₂O), Ferric Sulfate (FeSO₄.7H₂O), Magnesium Sulfate (MgSO₄7H₂O), Calcium Carbonate (CaCO₃), etc., would be used as the inorganiccompounds. If required, vitamins and base, etc., would be added. Theculture is performed for example, while shaking or aerating andagitating under aerobic condition, preferably at 20-40° C. for 5˜6 days.The pH of the medium preferably remains around neutrality. The5′-inosinic acid accumulated by direct fermentation is analyzed by theconventional method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0041] This invention will be better understood from the followingexamples. However, one skilled in the art will readily appreciate thespecific materials and the results described below are merelyillustrative of, and are not intended to, nor should be intended to,limit the invention as described more fully in the claims which followsthereafter.

EXAMPLE 1 Selection of microorganism sensitive to lysozyme (LY002)

[0042]Corynebacterium ammoniagenes strain CJ112 (ATCC-6872), the parentstrain, was suspended to 10⁷˜10⁸ cells/mL in the phosphate buffer (pH7.0) or citrate buffer (pH 5.5). N-methyl-N′-nitro-N-nitrosoguanidine(NTG) was added to a final concentration of 10˜50 μg/mL at roomtemperature or 32° C. After 20˜40 minutes, cells were washed twice with0.85% saline. Colonies were obtained by suitably suspending andspreading cells on a minimal medium (Medium 2) containing 1.7% agar.Then, each colony was tooth picked on to the minimal medium (Medium 2)containing 1.7% agar and on the minimal medium (Medium 2) containing1.7% agar and 40 pg/mL of lysozyme. First, microorganisms that grew onthe minimal medium containing 1.7% agar, but not on the minimal mediumcontaining 40 μg/mL of lysozyme were selected. The microorganism was tobe a parent strain and to be continuously induced in the above mutation.In a second screen, microorganisms that did not grow on the minimalmedium containing 16 μg/mL lysozyme were selected. Finally, according tothe above method, microorganisms that did not grow on the minimal mediumcontaining 8 μg/mL of lysozyme were selected. The colony of highlylysozyme sensitive microorganisms was cultivated on a nutrition medium(Medium 1), then cultivated for 24 hours on a seed medium (Medium 3),and cultivated for 3˜4 days on a culture medium (Medium 4) thereby toselect LY002 that can produce 5′-inosinic acid accumulated in theculture medium at the largest amounts. The concentration of lysozyme, atwhich the microorganism shows sensitivity, is listed in Table 2. TABLE 2CJ112 LY002 Concentration of lysozyme 80 μg/ml 8 μg/ml

EXAMPLE 2 Selection of Streptomycin-Resistant Strain (CISM10)

[0043] The mutant of Example 1, strain LY002, was used as the parentstrain for Example 2. LY002 was suspended to 10⁷˜10⁸ cells/mL in thephosphate buffer (pH 7.0) or citrate buffer (pH 5.5). NTG mutagenesiswas performed as in Example 1. Colonies were obtained by suitablysuspending and spreading cells on a 3 minimal media (Medium 2), whichcontained 1.7% agar and 1,000 μg/ml, 1,500 μg/mL or 2,000μg/mL ofstreptomycin, respectively. Then, each colony was cultivated on thenutrition medium (Medium 1), then cultivated for 24 hours on a seedmedium (Medium 3), and cultivated for 3˜4 days on a culture medium(Medium 4) thereby to select CISM10 that can produce 5′-inosinic acidaccumulated in the culture medium at the largest amounts. Theconcentration of streptomycin, at which the microorganism showsresistance, is listed in Table 3. TABLE 3 LY002 CISM10 Concentration ofstreptomycin 500 μg/ml 2,000 μg/ml

EXAMPLE 3 Selection of 3,4-dehydroproline-Resistant Strain (CS101)

[0044] The mutant of Example 2, strain CISM10, was used as the parentstrain for Example 3. CISM10 was suspended to 10⁷˜10⁸ cells/mL in thephosphate buffer (pH 7.0) or citrate buffer (pH 5.5). NTG mutagenesiswas performed as in Example 1. Colonies were obtained by suitablysuspending and spreading cells on a 3 minimal media (Medium 2), whichcontained 1.7% agar and 1,500 μg/ml, 2,500 μg/ml, or 3,500 μg/mL of3,4-dehydroproline, respectively. Then, each colony was cultivated onthe nutrition medium (Medium 1), then cultivated for 24 hours on a seedmedium (Medium 3), and cultivated for 3-4 days on a culture medium(Medium 4) thereby to select CIS104 that can produce 5′-inosinic acidaccumulated in the culture medium at the largest amounts. Theconcentration of 3,4-dehydroproline, at which the microorganism showsresistance, is listed in Table 4. TABLE 4 CISM10 CS101 Concentration of3,4-dehydroproline 1,000 μg/ml 3,500 μg/ml

EXAMPLE 4 Selection of L-azetidine-2-carboxylic Acid-Resistant Strain(CIAC12)

[0045] The mutant of Example 3, strain CS101, was used as the parentstrain for Example 4. CS101 was suspended to 10^(7˜10) ⁸ cells/mL in thephosphate buffer (pH 7.0) or citrate buffer (pH 5.5). NTG muta genesiswas performed as in Example 1. Colonies were obtained by suitablysuspending and spreading cells on a 3 minimal media (Medium 2), whichcontained 1.7% agar and 10 μg/ml, 20 μg/ml, or 30 μg/mL ofL-azetidine-2-carboxylic acid, respectively. Then, each colony wascultivated on the nutrition medium (Medium 1), then cultivated for 24hours on a seed medium (Medium 3), and cultivated for 3˜4 days on aculture medium (Medium 4), thereby to select CIAC12 that can produce5′-inosinic acid accumulated in the culture medium at the largestamounts. The concentration of L-azetidine-2-carboxylic acid, at whichthe microorganism shows resistance, is listed in Table 5. TABLE 5Property CISM10 CIAC12 Concentration of L-azetidine-2-carboxlic acid 5mg/ml 30 mg/ml

EXAMPLE 5 Selection of L-thiazolidine-4-carboxylic Acid-Resistant Strain(CITP13)

[0046] The mutant of Example 4, strain CIAC12, was used as the parentstrain for Example 5. CIAC12 was suspended to 10⁷˜10⁸ cells/mL in thephosphate buffer (pH 7.0) or citrate buffer (pH 5.5). NTG mutagenesiswas performed as in Example 1. Colonies were obtained by suitablysuspending and spreading cells on a 3 minimal media (Medium 2), whichcontained 1.7% agar and 20 μg/ml, 50 μg/ml, or 100 μg/mL ofL-thiazolidine-4-carboxylic acid, respectively. Then, each colony wascultivated on the nutrition medium (Medium 1), then cultivated for 24hours on a seed medium (Medium 3), and, cultivated for 3˜4 days on aculture medium (Medium 4) thereby to select CITP13 that can produce5′-inosinic acid accumulated in the culture medium at the largestamounts. The concentration of L-thiazolidine-4-carboxylic acid, at whichthe microorganism shows resistance, is listed in Table 6. TABLE 6 CISM10CS101 Concentration of L-thiazolidine-4-carboxylic acid 10 μg/ml 100μg/ml

EXAMPLE 6 Selection of Azaserine-Resistant Strain CJIPP009 (KCCM-10226)

[0047] The mutant of Example 5, strain CITP13, was used as the parentstrain for Example 6. CITP13 was suspended to 10⁷˜10⁸ cells/mL in thephosphate buffer (pH 7.0) or citrate buffer (pH 5.5). NTG mutagenesiswas performed as in Example 1. Colonies were obtained by suitablysuspending and spreading cells on a 3 minimal media (Medium 2), whichcontained 1.7% agar and 50 μg/ml, 75 μg/ml, or 100 μg/mL of azaserine,respectively. Then, each colony was cultivated on the nutrition medium(Medium 1), then cultivated for 24 hours on a seed medium (Medium 3),and cultivated for 3-4 days on a culture medium (Medium 4) thereby toselect CJIP009 (KCCM-10226) that can produce 5′-inosinic acidaccumulated in the culture medium at the largest amounts. Theconcentration of azaserine, at which the microorganism shows resistance,is listed in Table 7. TABLE 7 CITP13 CJIP009 (KCCM-10226) Concentrationof azaserine 25 μg/ml 100 μg/ml

EXAMPLE 7 Measurement of the Accumulation Amount of 5′-Inosinic Acid ina Smaller Scale Culture

[0048] Strain:

[0049] CJIP009 (KCCM-10226)

[0050] Seed Medium:

[0051] Glucose 5%, peptone 0.5%, Beef extract 0.5%, Yeast Extract 1%,Sodium Chloride (NaCl) 0.25%, Adenine 100 mg/L, Guanine 100 mg/L, pH7.2.

[0052] Flask Fermentation Medium:

[0053] Sodium Glutamate 0.1%, Ammonium Chloride (NH₄C1) 1.0%, MagnesiumSulfate (MgSO₄.7H₂O) 1.2%, Calcium Chloride (CaCl₂) 0.01%, FerricSulfate (FeSO₄.7H₂O) 20 mg/L, Manganese Sulfate (MnSO₄.H₂O) 20 mg/L,Zinc Sulfate (ZnSO₄ 7H₂O) 20 mg/L, Cupric Sulfate (CuSO₄ 7H₂O) 5.0 mg/L,L-Cystein 23 mg/L, Alanine 24 mg/L, Nicotinic acid 8.0 mg/L, .Biotin 45μg/L, Thiamine-HCl 5.0 mg/L, Adenine 30 mg/L, phosphoric acid(H₃PO₄)(85%) 1.9%, the mixture of Fructose, Glucose and molasses to 8%as reducing sugar (pH 7.2)

[0054] Fermentation Procedure:

[0055] 3 mL of the seed medium was introduced into a test tube of 18 mmdiameter and sterilized under elevated pressure by known methods. StrainCJIP009 was inoculated into the sterilized medium and cultivated whileshaking at 30° C. for 24 hours to use as the seed medium. 27 mL offermentation medium was introduced into 500 mL of Erlenmeyer flask forshaking and sterilized under elevated pressure at 120° C. for 10minutes. 3 mL of seed culture was then inoculated and cultivated for 5˜6days. The flask was shaken at 200 rpm at the temperature of 30° C. andpH of 7.2.

[0056] The amount of 5′-inosinic acid that accumulated was 19.1 g/L.

EXAMPLE 8 Measurement of the Accumulation Amount of 5′-Inosinic Acid ina Larger Scale Culture

[0057] Strain:

[0058] CJIP009 (KCCM-10226)

[0059] Seed medium: same as example 7

[0060] Fermentor Seed Medium

[0061] Glucose 5.4%, peptone 1.0%, Yeast Extract 2.0%, PotassiumDihydrogen Phosphate (KH₂PO₄) 0.1%, Potassium Monohydrogen Phosphate(K₂HPO₄) 0.1%, Magnesium Sulfate (MgSO₄.7H₂O) 0.1%, Ammonium Sulfate((NH4)₂SO₄) 0.5%, Ferric Sulfate (FeSO₄.7H₂O) 80 mg/L, Zinc Sulfate(ZnSO₄.7H₂O) 40 mg/L, Manganese Sulfate (MnSO₄.H₂O) 40 mg/L, L-Cystein80 mg/L Calcium Pantothenate 60 mg/L, Thiamine-HCl 20 mg/L, Biotin 240μg/L, Adenine 1200 mg/L, Guanine 1200 mg/l (pH 7.2)

[0062] Fermentor Main Medium

[0063] Calcium Chloride (CaCl₂) 120 mg/L, Cupric Sulfate (CuSO_(4˜7)H₂O)8.0 mg/L, Magnesium Sulfate (MgSO₄.7H₂O) 1.5%, Ferric Sulfate(FeSO₄.7H₂O) 24 mg/L, Zinc Sulfate (ZnSO₄.7H₂O) 24 mg/L, ManganeseSulfate (MnSO₄-H₂O) 24 mg/L, L-Cystein 26.4 mg/L, Sodium Glutamate0.12%, Thiamine-HCl 6.0 mg/L, Biotin 40 μg/L, Nicotinic acid 50 mg/L,Alanine 145 mg/L, Adenine 200 mg/L, phosphoric acid (H₃PO₄)(85%) 4.3%,the mixture of Fructose, Glucose and molasses to 32% as reducing sugar(pH 7.2)

[0064] Fermentation Procedure:

[0065] 50 mL of the seed medium was introduced into 500 mL of Erlenmeyerflask for shaking and sterilized under elevated pressure by the generalmethod. Strain CJIP009 was inoculated on the seed medium and cultivatedwhile shaking at 30° C. for 24 hours. The culture solution obtained wasused as a seed culture.

[0066] A liter (1000 mL) of seed medium was sterilized in a 2.5 Lfermentor under elevated pressure at 120° C. for 15 minutes. This mediumwas inoculated with 50 mL of the above seed culture and cultivated for1˜2 days while shaking at 900 rpm at the temperature of 28˜34° C. at apH of 7.2. The culture solution obtained was used as a main culture.

[0067] A 5 L fermentor was filled with 1,250 mL of fermentor main mediumand sterilized under elevated pressure at 120° C. for 15 minutes. Thismedium was inoculated with 250 mL of the above main culture andcultivated. When reducing sugars were present in the medium at aconcentration of 2%, a mixture of Fructose, glucose, and molasses wasadded four times until the final concentration of reducing sugars in theculture solution was increased to 32% (w/v). After a reducing sugarconcentration of 32% was achieved, the culture was cultivated for 5-6days while shaking at 900 rpm at the temperature of 30° C. and a pH of7.2.

[0068] The amount of 5′-inosinic acid that accumulated in the medium was70.3 g/L.

INDUSTRIAL APPLICABILITY

[0069] According to the present invention, 5′-inosinic acid can beobtained in a higher concentration and yield than in the prior art.According to the present invention, 5′-inosinic acid can be obtainedmore economically than in the prior art.

What is claimed is:
 1. Isolated Corynebacterium ammoniagenes strainCJIP009 having Accession No. KCCM-10226.
 2. The isolated Corynebacteriumammoniagenes strain CJIP009 of claim 1, wherein said strain is resistantto at least one L-glutamine analog selected from the group consisting ofazaserine and 6-diazo-5-oxo-L-norleucine. (DON).
 3. The isolatedCorynebacterium ammoniagenes strain CJIP009 of claim 1, wherein saidstrain is resistant to at least one L-proline analog selected from thegroup consisting of 3,4-dehydroproline, L-azetidine-2-carboxylic acid,L-thiazolidine-4-carboxylic acid, (S)-2,2-dimethyl-4-oxazolidecarboxylicacid, (S)-5,5-dimethyl-4-thiazolide carboxylic acid,(4S,2RS)-2-ethyl-4-thiazoline-carboxylic acid,(2S,4S)-4-hydroxy-2-pyrroline-carboxylic acid, 2-piperidinecarboxylicacid, and 2,5-pyrrolidinedione.
 4. The isolated Corynebacteriumammoniagenes strain CJIP009 of claim 1, wherein said strain is resistantto at least one L-glutamine analog selected from the group consisting ofazaserine and 6-diazo-5-oxo-L-norleucine (DON) and resistant to at leastone L-proline analog selected from the group consisting of3,4-dehydroproline, L-azetidine-2-carboxylic acid,L-thiazolidine-4-carboxylic acid, (S)-2,2-dimethyl-4-oxazolidecarboxylicacid, (S)-5,5-dimethyl-4-thiazolide carboxylic acid,(4S,2RS)-2-ethyl-4-thiazoline-carboxylic acid,(2S,4S)-4-hydroxy-2-pyrroline-carboxylic acid, 2-piperidinecarboxylicacid, and 2,5-pyrrolidinedione
 5. The isolated Corynebacteriumammoniagenes strain CJIP009 of claim 1, wherein said strain is resistantto streptomycin.
 6. The isolated Corynebacterium ammoniagenes strainCJIP009 of claim 1, wherein said strain requires exogenous adenine. 7.The isolated Corynebacterium ammoniagenes strain CJIP009 of claim 1,wherein said strain is guanine or xanthine leaky.
 8. The isolatedCorynebacterium ammoniagenes strain CJIP009 of claim 1, wherein saidstrain is sensitive to about 8 μg/mL lysozyme.
 9. A process forproducing 5′-inosinic acid comprising: cultivating Corynebacteriumammoniagenes strain CJIP009 having Accession No. KCCM-10226 underconditions that allow 5′-inosinic acid to be produced, wherein afermented culture media is formed and 5′-inosinic acid is produced. 10.The process of claim 9 further comprising isolating 5′-inosinic acidfrom the fermented culture media.
 11. The process of claim 9 whereincultivating comprises: cultivating said strain on a seed medium at about20° C. for about 24 hours; cultivating on a fermentor seed mediumcomprising a reducing sugar at an initial concentration more than about2% at from about 28° C. to about 34° C., about 900 rpm, and about pH 7.2for from about 1 day to about 2 days until the concentration of thereducing sugar falls below about 2%; and adding mixture of reducingsugars to the media to a final reducing sugar concentration of at leastabout 8% (w/v); wherein a fermented media comprising 5′-inosinic acid isproduced.
 12. The process of claim 11, wherein the mixture of reducingsugars comprises glucose, fructose, and molasses.
 13. The process ofclaim 11, wherein the mixture of reducing sugars is added to a finalreducing sugar concentration of at least about 32% (w/v).